Chemical Compositions and Enantiomeric Distributions of Foliar Essential Oils of Chamaecyparis lawsoniana (A. Murray bis) Parl, Thuja plicata Donn ex D. Don, and Tsuga heterophylla Sarg.

As part of our continuing interest in the essential oil compositions of gymnosperms, particularly the distribution of chiral terpenoids, we have obtained the foliar essential oils of Chamaecyparis lawsoniana (two samples), Thuja plicata (three samples), and Tsuga heterophylla (six samples) from locations in the state of Oregon, USA. The essential oils were obtained via hydrodistillation and analyzed by gas chromatographic techniques, including chiral gas chromatography—mass spectrometry. The major components in C. lawsoniana foliar essential oil were limonene (27.4% and 22.0%; >99% (+)-limonene), oplopanonyl acetate (13.8% and 11.3%), beyerene (14.3% and 9.0%), sabinene (7.0% and 6.5%; >99% (+)-sabinene), terpinen-4-ol (5.0% and 5.3%; predominantly (+)-terpinen-4-ol), and methyl myrtenate (2.0% and 5.4%). The major components in T. plicata essential oil were (−)-α-thujone (67.1–74.6%), (+)-β-thujone (7.8–9.3%), terpinen-4-ol (2.7–4.4%; predominantly (+)-terpinen-4-ol), and (+)-sabinene (1.1–3.5%). The major components in T. heterophylla essential oil were myrcene (7.0–27.6%), α-pinene (14.4–27.2%), β-phellandrene (6.6–19.3%), β-pinene (6.4–14.9%; >90% (−)-β-pinene), and (Z)-β-ocimene (0.7–11.3%). There are significant differences between the C. lawsoniana essential oils from wild trees in Oregon and those of trees cultivated in other geographical locations. The essential oil compositions of T. plicata are very similar, regardless of the collection site. There are no significant differences between T. heterophylla essential oils from the Oregon Coastal Range or those from the Oregon Cascade Range. Comparing essential oils of the Cupressaceae with the Pinaceae, there are some developing trends. The (+)-enantiomers seem to dominate for α-pinene, camphene, sabinene, β-pinene, limonene, terpinen-4-ol, and α-terpineol in the Cuppressaceae. On the other hand, the (−)-enantiomers seem to predominate for α-pinene, camphene, β-pinene, limonene, β-phellandrene, terpinen-4-ol, and α-terpineol in the Pinaceae.


Introduction
Chamaecyparis Spach is a genus in the Cupressaceae.The World Flora Online currently recognizes seven species of the genus [1] The genus Thuja L. (Cupressaceae) is represented by five taxa [4]: Thuja koraiensis Nakai (found in Jilin Province of China and in North and South Korea) [5], Thuja occidentalis L. (in eastern North America, the tree ranges from southeastern Canada, Minnesota, Michigan, and New England, south through the Appalachian Mountains) [6], Thuja plicata Donn ex.D. Don (two populations in western North America, a coastal population ranging from the Alaskan panhandle, coastal British Columbia south into coastal northern California, and an inland population found in the Rocky Mountains of British Columbia heading south to northern Idaho and western Montana) [7], Thuja standishii (Gordon) Carrière (native to Japan) [8], and Thuja sutchuenensis Franch.(native to Sichuan Province, China, but probably extinct in the wild due to deforestation) [9].The genus has been important to the traditional healthcare systems in its natural ranges [10,11].
Chamaecyparis lawsoniana (A.Murray bis) Parl., Cupressaceae (Port Orford cedar) is a large tree, around 50 m tall with a trunk up to 3 m in diameter [15].The foliage has a lacy feathery appearance with leaves that are overlapping and scalelike, 2-3 mm long; the bark is thick, silvery-brown, and furrowed (Figure 1) [16].The natural range of C. lawsoniana is limited to a small area of coastal Oregon into northern California (Figure 2) [17].It has become an important ornamental outside of its natural range, particularly in Europe.Previous essential oil analyses have been carried out on C. lawsoniana cultivated in Japan [18], Belgium [19], Egypt [20,21], Iran [22], Spain [23], and Greece [24].A purpose of the present study is to characterize the foliar essential oil of C. lawsoniana growing in its natural habitat in the Oregon Coastal Range.Thuja plicata Donn ex D. Don, Cupressaceae (western red cedar) is a large tree, growing up to 75 m tall with a trunk up to 5 m in diameter; the thick, fibrous, fissured bark is reddish-brown or grayish-brown; the foliage is displayed as flat, pendant sprays with overlapping scale-like leaves (Figure 3) [26].There are two separate ranges of T. plicata, a Coast-Cascade portion from southeastern Alaska (56 • 30 ′ N) to northwestern California (40 • 30 ′ N), and a Rocky Mountain section from British Columbia (54 • 30 ′ N) to Idaho and Montana (45 • 50 ′ N) (Figure 4) [26].
The heartwood of T. plicata has been shown to be a source of tropone monoterpenoids [27][28][29][30][31] and lignans [32][33][34][35][36][37][38][39][40], and the dilactone thujin [41], while the bark and aerial parts have yielded diterpenoid derivatives [42,43].There have been several investigations on the foliar essential oil compositions of T. plicata growing wild in western North America [44,45], cultivated in Poland [46,47], cultivated in Serbia [48], and growing wild in Idaho, USA [49].In addition, the volatiles from resin extracts of T. plicata cultivated in Czechia have been reported [50].In this work, we had the opportunity to collect T. plicata samples from the Cascade Range of Oregon, so an additional purpose of this study is to test the hypothesis that the T. plicata from Oregon, a separate population from those from Idaho, presents differences in essential oil composition.
Plants 2024, 13, 1325 4 of 28 Thuja plicata Donn ex D. Don, Cupressaceae (western red cedar) is a large tree, growing up to 75 m tall with a trunk up to 5 m in diameter; the thick, fibrous, fissured bark is reddish-brown or grayish-brown; the foliage is displayed as flat, pendant sprays with overlapping scale-like leaves (Figure 3) [26].There are two separate ranges of T. plicata, a Coast-Cascade portion from southeastern Alaska (56°30′ N) to northwestern California (40°30′ N), and a Rocky Mountain section from British Columbia (54°30′ N) to Idaho and Montana (45°50′ N) (Figure 4) [26].The heartwood of T. plicata has been shown to be a source of tropone monoterpenoids [27][28][29][30][31] and lignans [32][33][34][35][36][37][38][39][40], and the dilactone thujin [41], while the bark and aerial parts have yielded diterpenoid derivatives [42,43].There have been several investigations on the foliar essential oil compositions of T. plicata growing wild in western North America [44,45], cultivated in Poland [46,47], cultivated in Serbia [48], and growing wild in Idaho, USA [49].In addition, the volatiles from resin extracts of T. plicata cultivated in Czechia have been reported [50].In this work, we had the opportunity to collect T. plicata samples from the Cascade Range of Oregon, so an additional purpose of this study is to test the hypothesis that the T. plicata from Oregon, a separate population from those from Idaho, presents differences in essential oil composition.
Tsuga heterophylla Sarg.(western hemlock) is a tree that grows up to 50 m tall with a trunk diameter up to 2 m; its leaves are needles, 5-20 mm long and 1.5-2 mm wide; its cones are small, 15-25 mm long and 10-25 mm wide; its bark is grey-brown, scaly, and moderately fissured (Figure 5) [51].The native range of T. heterophylla is from the coast of southern Alaska, south through coastal British Columbia, Washington, Oregon, and into coastal northern California (Figure 6) [52].The coastal range of T. heterophylla divides into an Oregon Coastal Range and a Cascade Range in Oregon.There is also a Rocky Mountain population that ranges from British Columbia south to northern Idaho and northwestern Montana (Figure 6).Tsuga heterophylla Sarg.(western hemlock) is a tree that grows up to 50 m tall with a trunk diameter up to 2 m; its leaves are needles, 5-20 mm long and 1.5-2 mm wide; its cones are small, 15-25 mm long and 10-25 mm wide; its bark is grey-brown, scaly, and moderately fissured (Figure 5) [51].The native range of T. heterophylla is from the coast of southern Alaska, south through coastal British Columbia, Washington, Oregon, and into coastal northern California (Figure 6) [52].The coastal range of T. heterophylla divides into an Oregon Coastal Range and a Cascade Range in Oregon.There is also a Rocky Mountain population that ranges from British Columbia south to northern Idaho and northwestern Montana (Figure 6).Extracts of the wood of T. heterophylla have yielded lignans, including matairesinol [53], 8-hydroxy-α-conidendrin, 8-hydroxy-α-conidendric acid methyl ester [54], and 8hydroxyoxomatairesinol [55].Foliar volatiles have also been examined [56][57][58].The purpose of the current study is to obtain foliar essential oils of T. heterophylla from both the Oregon Coastal Range and the Oregon Cascades to compare essential oil compositions from the two separated populations as well as to compare with compositions previously reported from British Columbia, Canada.
The essential oil compositions of the Oregon T. plicata samples were very similar to those from our previous collection from northern Idaho [49] as well as those reported by von Rudloff and co-workers [44], Tsiri and co-workers [46], Lis and co-workers [47], and Nikolić and co-workers [48].Indeed, a hierarchical cluster analysis (HCA) reveals very high similarity between the samples (Figure 9).The cluster analysis shows the greatest similarity, not surprisingly, between the samples from western North America (>99.94%similarity).Even the samples cultivated in Poland [47] showed >99.59% similarity to the North American samples.In comparing the Oregon samples from this work with those of our previous investigation of samples from Idaho, the concentrations of the major components are not statistically different (t-test, p > 0.05) (Figure 10).However, the βthujone t-test showed a p-value of 0.057.In retrospect, the similarities in essential oil compositions are consistent with the genomic analysis of T. plicata; there is little genetic differentiation in this species [64][65][66].The T. plicata samples were subjected to principal component analysis (PCA) to understand their chemical variability comprehensively.The results, which are highly precise, revealed that F1 and F2 accounted for a significant 99.99% of the entire chemical variability.This analysis effectively grouped the samples into four main categories, as illustrated in Figure 11.F1 demonstrated a positive correlation with α-thujone (6.695), while terpinen-4-ol (-2.430), sabinene (-2.344), and β-thujone (-1.921), showed negative correlations.On the other hand, F2 had a positive correlation with sabinene (0.206), but a negative correlation only with β-thujone (-0.170).Notably, the samples collected in Oregon and those from Idaho and von Rudloff were found to have similar chemical characteristics, with α-thujone concentrations close to 70% and sabinene amounts of less than 5.0%.The T. plicata samples were subjected to principal component analysis (PCA) to understand their chemical variability comprehensively.The results, which are highly precise, revealed that F1 and F2 accounted for a significant 99.99% of the entire chemical variability.This analysis effectively grouped the samples into four main categories, as illustrated in Figure 11.F1 demonstrated a positive correlation with α-thujone (6.695), while terpinen-4-ol (−2.430), sabinene (−2.344), and β-thujone (−1.921), showed negative correlations.On the other hand, F2 had a positive correlation with sabinene (0.206), but a negative correlation only with β-thujone (−0.170).Notably, the samples collected in Oregon and those from Idaho and von Rudloff were found to have similar chemical characteristics, with α-thujone concentrations close to 70% and sabinene amounts of less than 5.0%.[44], ID = samples from Idaho [49], OR = samples from Oregon (this work), Tsiri [46], Nikolic [48], Lis [47].
A hierarchical cluster analysis (HCA) was carried out to visualize the similarities between the T. heterophylla essential oil compositions (Figure 12).The HCA shows that the British Columbia samples and the Oregon samples #1-#3 and #5 form a relatively large cluster with >88% similarity.Oregon Coastal Range samples #4 and #6 are qualitatively similar to the large cluster, but different (with 66% similarity) in that sample #4 showed a lower myrcene concentration (only 7.0%), while sample #6 showed a relatively low βpinene concentration (6.4%); both samples were also low in β-phellandrene (6.6% and 8.4%, respectively).Curiously, a sample collected in 2020 from a single tree growing in the Hoyt Arboretum near Portland, Oregon, was very different in composition with only 5.7% monoterpene hydrocarbons, including no observed α-pinene [58].The concentration of α-terpineol (10.3%) was relatively high in the Hoyt Arboretum sample.It is not clear what factors may account for the dissimilarity between the Hoyt Arboretum sample and the other T. heterophylla samples.The Hoyt Arboretum sample was collected in September 2020, while the samples in this present study were collected in April 2023.However, von Rudloff sampled trees from Vancouver, British Columbia, in both March 1974, and October 1974, which showed no significant difference in the α-pinene concentrations (both 15.3%) or the α-terpineol concentrations (0.8% and 0.5%, respectively) [56].
In comparing the compositions of the samples from the Oregon Cascade and Coastal ranges, there are no significant differences between their major components (α-pinene, β-pinene, myrcene, α-phellandrene, limonene, β-phellandrene, (Z)-β-ocimene, benzoic acid, and beyerene) (Figure 13).This result is consistent with the previous study by von Rudloff [56], who found no significant differences in the compositions of trees located in the British Columbia Coastal Range and those of trees from the Rocky Mountains.The low level of genetic diversity can be explained by past vegetation history.That is, genetic diversity in T. heterophylla, as well as T. plicata, is likely to be diminished due to a population bottleneck during the last glacial maximum [67].
Based on this current work and previous studies of enantiomeric distributions of chiral monoterpenoids in conifer essential oils, there are some interesting trends (Table 7).(+)-α-Pinene is the dominant enantiomer in essential oils of the Cupressaceae, but, although it is not consistent, (−)-α-pinene generally predominates in the Pinaceae.Similar trends are seen for camphene, β-pinene, and limonene; the (−)-enantiomers are dominant in the Pinaceae while the (+)-enantiomers dominate the essential oils of the Cupressaceae.Although (+)-sabinene seems to be virtually exclusive in the Cupressaceae, the enantiomeric distribution is inconsistent in the Pinaceae.(−)-β-Phellandrene is clearly dominant in Pinaceae essential oils, but there are insufficient data to draw a conclusion regarding the Cupressaceae.(−)-Terpinen-4-ol and (−)-α-terpineol are slightly favored in the Pinaceae while the (+)-enantiomers are slightly favored in the Cupressaceae.There are not enough data regarding the enantiomeric distributions of linalool to draw a conclusion regarding the distribution trend.

Plant Material
The foliage of C. lawsoniana was collected from two separate trees (C.lawsoniana #1 and #2) on 15 April 2023, from the Van Duzer Forest, Oregon Coastal Range.The trees were identified in the field by W.N. Setzer using a field guide [16] and were verified through a comparison with samples from the New York Botanical Garden [74].A voucher specimen (WNS-Cl-6886) has been deposited into the herbarium at the University of Alabama in Huntsville.The fresh foliage from each tree was frozen (−20 • C) and stored frozen until distillation.Foliage of T. plicata was collected from three different individual trees (T.plicata #1-#3) located near Mt.Hood Village, Oregon, on 14 April 2023 (Table 8).The trees were identified by W.N. Setzer [16,75] and a voucher specimen (WNS-Tp-6850) has been deposited into the herbarium at the University of Alabama in Huntsville.The fresh foliage was immediately frozen and stored frozen (−20 • C) until distillation.Tsuga heterophylla foliage from three individual trees (T.heterophylla #1-#3) was collected on 14 April 2023 near Mt.Hood Village, Oregon (Cascade Range) and from three individual trees (T.heterophylla #4-#6) on 16 April 2023 near Ross Lodge-Boger, Oregon (Coastal Range) (Table 8).The trees were identified in the field by W.N. Setzer using a field guide [16] and were verified through a comparison with botanical samples from the C. V. Starr Virtual Herbarium [76].A voucher specimen, WNS-Th-6897, has been deposited into the herbarium at the University of Alabama in Huntsville.The foliage was frozen (−20 • C) and stored frozen until hydrodistillation.

Hydrodistillation
The fresh/frozen foliage of each sample was chopped and hydrodistilled for three hours using a Likens-Nickerson apparatus [77][78][79] with the continuous extraction of the distillate with dichloromethane (Table 8).Enough water to immerse the plant material was used for the hydrodistillation.The condenser was chilled (10-15 • C) using a refrigerated recirculating pump.Each plant sample was hydrodistilled once.The dichloromethane was evaporated using a stream of warm air.

Gas Chromatographic Analysis
The C. lawsoniana, T. plicata, and T. heterophylla foliar essential oils were analyzed via GC-MS, GC-FID, and chiral GC-MS as previously described [73].The essential oil compositions were determined by comparing both MS fragmentation and RI values with those reported in the Adams [60], FFNSC3 [61], NIST20 [62], and Satyal [63] databases.The percent compositions were determined from raw peak areas (GC-FID) without standardization.Enantiomeric distributions were determined via the comparison of RI values with authentic samples (Sigma-Aldrich, Milwaukee, WI, USA), which were compiled in our in-house database.
Principal component analysis (PCA), type Pearson correlation, was carried out to verify the previous HCA analysis using the main essential oil components (as described above).The PCA analyses were carried out using XLSTAT v. 018.1.1.62926(Addinsoft, Paris, France).

Conclusions
The present work revealed that wild-growing native Chamaecyparis lawsoniana essential oils show significant differences compared to the essential oils from trees cultivated in other geographical locations.On the other hand, essential oils of Thuja plicata are very similar, regardless of the collection site.Likewise, there are no significant differences between the Tsuga heterophylla essential oils from the Oregon Coastal Range and those from the Oregon Cascade Range.Both T. plicata and T. heterophylla likely have diminished genetic diversity, likely due to population bottlenecks during the last ice age.An examination of the distribution of monoterpenoid enantiomers indicates that the (+)-enantiomers seem to dominate α-pinene, camphene, sabinene, β-pinene, limonene, terpinen-4-ol, and αterpineol in the Cuppressaceae, while the (−)-enantiomers seem to predominate for α-pinene, camphene, β-pinene, limonene, β-phellandrene, terpinen-4-ol, and α-terpineol in the Pinaceae.It would be interesting to see if these trends in enantiomeric distributions continue with additional research on the essential oils of gymnosperms.

Plants 2024 , 31 Figure 1 .
Figure 1.Chamaecyparis lawsoniana (A.Murray bis) Parl.(A): A photograph of its foliage, (B): A photograph of its bark (photographs taken by K. Swor at the time of sample collection).

Figure 1 .
Figure 1.Chamaecyparis lawsoniana (A.Murray bis) Parl.(A): A photograph of its foliage, (B): A photograph of its bark (photographs taken by K. Swor at the time of sample collection).

Figure 1 .
Figure 1.Chamaecyparis lawsoniana (A.Murray bis) Parl.(A): A photograph of its foliage, (B): A photograph of its bark (photographs taken by K. Swor at the time of sample collection).

Figure 2 .
Figure 2. Natural range of Chamaecyparis lawsoniana [25].This image is in the public domain in the United States because it only contains materials that originally came from the United States Geological Survey, an agency of the United States Department of the Interior.

Figure 2 .
Figure 2. Natural range of Chamaecyparis lawsoniana [25].This image is in the public domain in the United States because it only contains materials that originally came from the United States Geological Survey, an agency of the United States Department of the Interior.

Figure 3 .
Figure 3. Photographs of Thuja plicata taken by K. Swor at the time of sample collection.(A): A photograph of its foliage.(B): A photograph of its bark.

Figure 3 . 31 Figure 4 .
Figure 3. Photographs of Thuja plicata taken by K. Swor at the time of sample collection.(A): A photograph of its foliage.(B): A photograph of its bark.Plants 2024, 13, x FOR PEER REVIEW 5 of 31

Figure 4 .
Figure 4.The native range of Thuja plicata [25].This image is in the public domain in the United States because it only contains materials that originally came from the United States Geological Survey, an agency of the United States Department of the Interior.

Figure 6 .
Figure 6.The native range of Tsuga heterophylla [25].This image is in the public domain in the United States because it only contains materials that originally came from the United States Geological Survey, an agency of the United States Department of the Interior.

Figure 6 .
Figure 6.The native range of Tsuga heterophylla [25].This image is in the public domain in the United States because it only contains materials that originally came from the United States Geological Survey, an agency of the United States Department of the Interior.

Figure 6 .
Figure 6.The native range of Tsuga heterophylla [25].This image is in the public domain in the United States because it only contains materials that originally came from the United States Geological Survey, an agency of the United States Department of the Interior.

Table 1 .
The foliar essential oil composition (%) of Chamaecyparis lawsoniana from the Oregon Coastal Range.

Table 2 .
The foliar essential oil composition (percentages) of Thuja plicata from the Cascade Range, Oregon.

Table 2 .
The foliar essential oil composition (percentages) of Thuja plicata from the Cascade Range, Oregon.

Table 3 .
The foliar essential oil compositions (percentages) of the Tsuga heterophylla from the Oregon Cascade Range and the Oregon Coastal Range.

Table 4 .
The enantiomeric distribution (percent of each enantiomer) of the chiral terpenoids in Chamaecyparis lawsoniana.RI db = Retention index from our in-house database based on commercially available compounds available from Sigma-Aldrich and augmented with our own data.RI calc = Calculated retention index based on a series of n-alkanes on a Restek B-Dex 325 capillary column.n.o.= not observed.n.a.= no reference compound available.

Table 5 .
The enantiomeric distribution (percent of each enantiomer) of the chiral terpenoids in Thuja plicata.Retention index from our in-house database based on commercially available compounds available from Sigma-Aldrich and augmented with our own data.RI calc = Calculated retention index based on a series of n-alkanes on a Restek B-Dex 325 capillary column.n.o.= not observed.n.a.= no reference compound available.

Table 6 .
The enantiomeric distribution (percent of each enantiomer) of the chiral terpenoid components in the foliar essential oil of the Tsuga heterophylla from Oregon.Retention index from our in-house database based on commercially available compounds available from Sigma-Aldrich and augmented with our own data.RI calc = Calculated retention index based on a series of n-alkanes on a Restek B-Dex 325 capillary column.n.o.= not observed.n.a.= no reference compound available.

Table 7 .
The enantiomeric distribution (percent of each enantiomer) of the chiral terpenoids in members of the Pinaceae and Cupressaceae.
a Enantiomer misassigned in the original report.n.o.= not observed.t.w.= this work (mean values).